The goal of this research is to determine the mechanism and regulation of the initiation of DNA replication in eukaryotic cells. It is clear that for maintenance of the integrity of the genome from one cell generation to the next, DNA must be duplicated in a highly controlled and accurate manner. Interruption of these controls may promote genome instability and lead to neoplastic transformation. Moreover, the DNA replication proteins represent tangible targets for therapeutic intervention and diagnosis of proliferation of cancer cells, and other proliferative disorders. The initiator protein (ORC) cooperates with a series of DNA replication proteins to establish at origins of DNA replication a complex that allows later initiation of DNA synthesis at each origin. The initiator protein is a multi-subunit, sequence-specific DNA binding protein but it may have functions in other aspects of the chromosome inheritance cycle. The proposed research in this application will investigate how DNA replication occurs in an ORC- and origin-dependent manner and will study how initiation of DNA replication is temporally controlled throughout the cell division cycle. In specific aim 1, the formation of a pre-replication complex will be investigated. In specific aim 2, proteins that interact with the mini-chromosome maintenance complex, such as Cdc45 will be studied, as will modifications of the Mcm4 proteins, in specific aim 3, the potential role of Orc6 in chromosome segregation and cytokinesis will be explored. Specific aim 4 has a goal of determining the role of dNTP levels in regulation of DNA replication. [unreadable] [unreadable]